Project Abstract/ Summary Non-alcoholic fatty liver disease (NAFLD) affects between 80 to 100 million US citizens. The most common form of the disease is a benign and non-progressive condition called non-alcoholic fatty liver (NAFL). However, ~20% of NALFD cases are classified as non-alcoholic steatohepatitis (NASH), an inflammatory condition that commonly progresses to liver fibrosis, cirrhosis, and finally to liver failure or liver cancer. The only way to differentiate NAFL from NASH is via invasive biopsy which is expensive, poses a non-negligible risk of serious internal bleeding, and is thus ill-suited for patient follow-up. Biopsy is not a practical screening tool and as a result NASH typically goes undiagnosed until progression to advanced disease. There is a survival benefit to early diagnosis and a number of NASH therapeutics are anticipated within the next few years. Non-invasive methods to either diagnose NASH, or to monitor treatment response are sorely needed. There have been advances toward radiologic diagnosis of advanced fibrosis and cirrhosis but there are no methods to image the liver inflammation that invariantly precedes disease progression. Myeloperoxidase (MPO) is a potential imaging biomarker for differential diagnosis of NASH. MPO is secreted by activated neutrophils as part of the inflammatory microenvironment and converts reactive oxygen species (ROS) generated by neutrophil respiration into highly injurious ROS like perchlorous acid. MPO is highly abundant in the extracellular space of inflamed liver, but is scarce in healthy tissue. We recently developed a small molecule Fe-based MRI contrast agent (Fe-PyC3A) that undergoes a 10-fold increase in relaxivity (MR signal generating potency) in the presence of ROS. This unprecedentedly large relaxivity change is achieved by oxidation of Fe2+ to Fe3+. The Fe2+ ion is a very inefficient relaxation agent but high-spin Fe3+ is a very potent relaxation agent. Preliminary imaging experiments using a mouse model of acute pancreatitis demonstrate that Fe-PyC3A provides little-to-no contrast enhancement of healthy tissue but strong and selective contrast-enhancement of acutely inflamed tissue. Importantly, the level of contrast enhancement correlates significantly and positively with MPO activity levels determined by ex vivo laboratory quantitation (r = 0.95, P < 0.0001). This proposal focuses on optimizing and validating redox active Fe complexes as MPO-specific contrast agents for liver MRI. The ultimate goal of this research is a non-invasive imaging test to differentiate patients with non-progressive NAFL from patients will benefit from invasive biopsy. The immediate outputs of the proposed work will be an MRI contrast agent that is 1) specific to acute inflammation, 2) can differentiate inflamed vs. non-inflamed fatty liver in mice, and 3) does not exhibit pre-clinical safety signals.